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* Department of Environmental Medicine and the Lung Biology and Disease Program;
Cardiovascular Research Institute and Department of Medicine; and
Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, USA
1Correspondence: Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Box 850, 601 Elmwood Ave., Rochester, NY 14642, USA. E-mail: irfan_rahman{at}urmc.rochester.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidants • eNOS • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. VEGF-VEGFR-2-mediated neovascularization plays an important role in maintaining the normal lung structure (2)
. Several lines of investigation reveal that inhibition of the VEGF-VEGFR-2 signaling pathway results in decreased lung alveolarization and arterial density (3
, 4)
. Lung microvasculature is critical for gas exchange and alveolar integrity. Emphysema, one of the major features of chronic obstructive pulmonary disease (COPD), is defined as enlargement of the distal air spaces as a consequence of alveolar wall destruction. Cigarette smoke (CS) is a complex mixture of >4700 chemical compounds and contains a high concentration of free radicals (>1017 molecules/puff), which is known to induce oxidative stress and inflammation in the lung (5)
. CS-induced oxidative stress is involved in the development of emphysema, and CS-induced emphysematous alveolar septa are remarkably thin and almost avascular (6
, 7)
. In addition, CS also plays an important role in the etiology of cardiovascular diseases and vascular dysfunction, such as atherosclerosis and pulmonary hypertension (8
9
10
11
12)
. This is due to the fact that CS induces endothelial dysfunction in the systemic and pulmonary circulation leading to the earliest morphological and pathological changes in the vessel wall (12
13
14)
. However, the mechanisms by which CS impairs endothelial function, including cell survival, proliferation, and angiogenesis, remain unknown.
Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining vascular integrity (15)
and is activated by VEGF and shear stress through the VEGFR-2 (16
17
18)
. It is also known that nitric oxide (NO) is an essential mediator of endothelial cell migration and VEGF-induced angiogenesis (19)
. VEGF acts via its receptors VEGFR-1 (Flt1) and VEGFR-2 (Flk-1/KDR) to induce vascular permeability, cell proliferation, cellular migration, and tube formation in endothelial cells (1)
. Both VEGF and VEGFR-2 expression levels are reduced in emphysematous lungs (20
, 21)
. We have recently shown that both VEGF and VEGFR-2 expression are reduced in response to CS exposure in rat lungs (22)
. Recent studies (21)
reported that chronic inhibition of global VEGF receptors using SU5416 in rats caused airway enlargement and alveolar endothelial cell death and decreased arterial density, suggesting that angiogenesis is necessary for alveolarization. However, the role of VEGFR-1 and VEGFR-2 in regulation of CS-induced alteration in alveolarization was not studied. It was postulated that oxidative stress plays a central role in alveolar septal destruction as a result of VEGFR inhibition, as shown by the preventive role of a superoxide dismutase mimetic (M40419) administrated in combination with VEGFR inhibitor (SU5416) in rat lung (23)
. The SU5416 compound inhibits both VEGFR-1 and VEGFR-2 kinases (21)
, whereas NVP-AAD777 is a specific inhibitor of VEGFR-2 kinase (22)
. The effect of a specific VEGFR-2 inhibitor on CS-mediated oxidative stress, inflammation, and endothelial dysfunction has not been evaluated in any in vitro or in vivo models. We hypothesized that specific inhibition of VEGFR-2 causes oxidative stress and an inflammatory response, which lead to endothelial dysfunction, and that these responses are augmented by CS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VEGFR-2 inhibitor treatment and CS exposure
The animal protocol was approved by the University Committee on Animal Resources (University of Rochester, Rochester, NY, USA). Male C57BL/6J mice, 8-wk-old, 25–30 g (Jackson Laboratory, Bar Harbor, ME, USA), were divided into four exposure groups (n=8): 1) air + vehicle; 2) CS + vehicle; 3) air + VEGFR-2 inhibitor [NVP-AAD777 (IC50=510 nM), Novartis Pharma AG, Basel, Switzerland]; and 4) CS+VEGFR-2 inhibitor. The VEGFR-2 inhibitor, NVP-AAD777 was dissolved in polyethylene glycol-400 (PEG 400) as a vehicle and administered at 20 mg/kg body weight subcutaneously, 3x/wk as described previously (21
, 22)
. The details of VEGFR-2 kinase inhibition using this compound are given in our previous studies (22)
. Control animals were administered vehicle alone in the same manner. Mice were placed inside a ventilated chamber and exposed to whole body CS using Baumgartner-Jaeger CSM 2072i smoking machine (CH Technologies, Westwood, NJ, USA) at 300 mg/m3 total particulate matter (TPM) for 2 h/day (1 h apart 2x/day), 5 days/wk for 3 wk with 2R4F research grade cigarettes (University of Kentucky, Lexington, KY, USA; refs. 24
, 25
). The level of carbon monoxide in the chamber was 350 ppm. Mice were sacrificed 2 h after last exposure by intraperitoneal injection of 50 mg/kg sodium pentobarbital. The level of carboxyhemoglobin in blood was 17%. Control mice were exposed to filtered air in an identical chamber according to the same protocol described for CS exposure.
Lung fixation for morphology
Mouse lungs (4 lungs from each group) were inflated with 4% paraformaldehyde at a constant pressure of 25 cm. The inflated lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 µm) were obtained and used for morphological analyses.
Inflammatory cell counts in bronchoalveolar lavage fluid (BALF)
Lungs were lavaged via a butterfly cannula inserted into the trachea and instilled with saline. Cells were separated by centrifugation of BALF and resuspended in 1 ml of saline. Differential cell counts (minimum of 500 cells per slide) were performed on cytospin-prepared slides (Thermo Shandon, Pittsburgh, PA, USA) stained with Diff-Quick (Dade Behring, Newark, DE, USA).
Immunoblotting in lung tissue homogenate
Frozen lung tissues (0.1 g) were homogenized in 0.5 ml of ice-cold radio-immuno-precipitation assay (RIPA) lysis buffer containing complete protease inhibitor cocktail (Sigma-Aldrich). The homogenates were kept on ice for 45 min and then centrifuged at 10,000 g for 15 min at 4°C, and the supernatants were separated. Protein levels were measured using bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). Protein (30 µg) was electrophoresed on a 4–15% gradient PAGE gel and transblotted onto nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked with 5% nonfat milk and incubated with relevant primary antibody (1:1000 dilution). Primary antibodies used were mouse anti-phosphotyrosine clone 4G10 (Upstate Cell Signaling, Temecula, CA, USA), rabbit anti-VEGFR-2, rabbit anti-eNOS (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed, bound antibody was detected using anti-rabbit/mouse antibody (1:20,000 dilution) linked to horseradish peroxidase, and bound complexes were detected using enhanced chemiluminescence (Perkin Elmer, Waltham, MA, USA).
eNOS immunoprecipitation
Mouse lung homogenates were prepared in RIPA buffer as described above, and eNOS protein was immunoprecipitated using anti-eNOS antibody (1:100 dilution; rabbit anti-eNOS, Cell Signaling Technology), which was added to 250 µg of protein in a final volume of 400 µl and incubated for 1 h. Protein-A/G agarose beads (20 µl; Santa Cruz Biotechnology) were added to each sample and left overnight at 4°C on a rocker. The samples were then centrifuged at 10,000 g at 4°C for 5 min. The supernatant was discarded, and the beads were washed 3x and then resuspended in 100 µl of RIPA buffer. The samples were then mixed with 5x SDS sample buffer and boiled, and the proteins were resolved by SDS-PAGE. Immunoblotting was performed for phosphorylated (p) -eNOS [rabbit anti-p-(Ser-1177) eNOS, Cell Signaling Technology] and e-NOS (rabbit anti-eNOS, Cell Signaling Technology).
Cytokines and VEGF analysis in lung tissue homogenate by the Luminex method
Whole lung tissues were homogenized in ice-cold buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol; and 0.5 mM PMSF) and incubated with Nonidet P-40 for 15 min. After 15 min, samples were centrifuged, and the supernatant was used to analyze the levels of proinflammatory mediators, [neutrophil chemotactic factor (KC)/monocyte chemoattractant protein-1 (MCP-1), KC/CXCL1, MCP-1/CCL2, interleukin (IL) -6, and tumor necrosis factor (TNF) -
] and VEGF levels in lungs using a Luminex-based assay (mouse multicytokine beadmaster beadlyte kit, Upstate Cell Signaling) as instructed by the manufacturer. Data were obtained using a Luminex 100 reader (Luminex Corporation, Austin, TX, USA). Protein levels were measured with a BCA kit (Pierce).
Immunohistochemistry
Paraffin was removed from paraffin-embedded lung sections and rehydrated before immunohistochemistry. The sections were refixed, and immunostaining was performed using an antibody specific for 4-hydroxy-2-nonenal (4-HNE) adducts (anti-4-HNE antibody; OXIS International, Foster City, CA, USA) or macrophages (Mac-3 antibody, BD Pharmingen, San Jose, CA, USA). Immunodetection was performed with a biotinylated rabbit anti-mouse Ig reagent (Dako, Carpinteria, CA, USA), streptavidin-biotin complex reagent (Dako), and 3',3-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Counterstaining was performed with hematoxylin (22)
. Macrophages in each lung section (10 lung sections from different regions of the lung) were counted under the microscope (x200) in a blinded manner, and the cell numbers were averaged. The intensity of 4-HNE adducts staining was assessed by scoring method in a blinded manner (0=no staining, 1=weak staining, 2.5=moderate staining, and 5=intense staining).
In vitro studies using human lung microvascular endothelial cells (HMVEC-Ls)
HMVEC-Ls were purchased from Cambrex (Walksville, MD, USA) and were grown in endothelial growth medium-2 (EGM-2; Cambrex) media containing 10% fetal bovine albumin (FBS) at 37°C in a humidified atmosphere containing 5% CO2. Cells were grown in 75 mm flasks coated with 0.1% gelatin, and treatments were performed in 0.1% gelatin-coated 6-well plates. Cigarette smoke extract (CSE) and VEGFR-2 inhibitor treatments were performed after cells were starved for 6 h with EGM-2 media containing 0.1% FBS.
CSE preparation
Research grade cigarettes (1R3F) were obtained from the Kentucky Tobacco Research and Development Center at the University of Kentucky. The composition of 1R3F research grade cigarettes was as follows: total particulate matter, 17.1 mg/cigarette; tar, 15 mg/cigarette; and nicotine, 1.16 mg/cigarette. CSE (10%) was prepared by bubbling smoke from one cigarette into 10 ml of EGM-2 culture media without FBS at a rate of 1 cigarette every 2 min, as described previously (26
, 27)
, using a modification of the method described by Carp and Janoff (28)
. The pH of the CSE was adjusted to 7.4, and it was sterile filtered through a 0.45 µm filter (Acrodisc; Pall Corporation, Ann Arbor, MI, USA). CSE preparation was standardized by measuring the absorbance (optical density=0.86±0.05) at a wavelength of 320 nm. The pattern of absorbance (spectrogram) observed at 
320 showed very little variation between different preparations of CSE. CSE was freshly prepared for each experiment and diluted with culture media supplemented with 0.1% FBS immediately before use. Control medium was prepared by bubbling air through 10 ml of culture media without FBS, the pH was adjusted to 7.4, and the medium was sterile filtered as described above.
Effect of CSE and VEGFR-2 inhibitor on reactive oxygen species (ROS) production in HMVEC-Ls
ROS production in HMVEC-Ls in response to CSE and VEGFR-2 inhibitor was assessed using a fluorescent dye, 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA; Invitrogen, Carlsbad, CA, USA). Inside the cell, the probe is deacetylated by esterases and forms H2DCF, which is oxidized by ROS to DCF, a highly fluorescent compound. Thus, dye oxidation is an indirect measure of the presence of ROS intermediates. HMVEC-Ls were grown in 0.1% gelatin-coated 8-chamber culture slides (BD Falcon, Bedford, MA, USA) and treated with CSE (0.1–0.5%), VEGFR-2 inhibitor (0.5–1.0 µM), and CSE (0.25%) + VEGFR-2 inhibitor (0.5 µM) for 2 h and washed twice with PBS. Cells were then incubated with 10 µM of carboxy-H2DCFDA for 15 min at room temperature and rinsed with PBS. The cells were observed under a fluorescent microscope, and the green fluorescent intensity was assessed using a scoring system (1=weak staining, 2=moderate staining, and 5=intense staining).
Effect of CSE on VEGFR-2 and eNOS protein expression
HMVEC-Ls were seeded in 6-well plates containing culture media supplemented with 10% FBS in a final volume of 2 ml. The cells were grown to 
90% confluent and starved in EGM-2 media containing 0.1% FBS for 6 h before the CSE treatments. All treatments were performed in duplicate. The cells were treated with CSE (0.1, 0.25, and 0.5%) for 12 h at 37°C in a humidified atmosphere containing 5% CO2. After a 12 h treatment, cell lysates were prepared in RIPA buffer containing a protease inhibitor cocktail to investigate the level of VEGFR-2 and e-NOS protein expression. Protein concentrations were analyzed using the BCA kit (Pierce). The levels of VEGFR-2 and eNOS were assessed using immunoblotting method with a rabbit anti-VEGFR-2 antibody (Cell Signaling Technology) and rabbit anti-eNOS antibody (BD Bioscience, San Jose, CA, USA), respectively.
Determination of the effect of CS on VEGF-induced VEGFR-2 phosphorylation
HMVEC-Ls were grown (90% confluent) in 6-well culture plates and starved for 6 h in 0.1% serum and VEGF-free media. Cells were then treated with CSE (0.1–0.5%) and VEGFR-2 inhibitor (0.5 and 1.0 µM; IC50=0.51 µM) for 2 h. A stock solution of NVP-AAD777 (1 mM) was prepared in 20 µl of DMSO. Therefore, some groups were treated with DMSO (0.02 µl/ml) as controls. At the end of the 2 h period, VEGF (50 ng/ml, Cell Signaling Technology) was added and incubated for 10 min at 37°C in a humidified atmosphere containing 5% CO2. Parallel control experiments were carried out without adding VEGF. The reaction was stopped immediately by adding ice-cold PBS, and the cells were washed twice with ice-cold PBS and then lyzed using RIPA buffer containing protease inhibitor cocktail. Finally, cell lysates were sonicated for 10 s and centrifuged at 10,000 g for 15 min, and the supernatant was separated and analyzed for protein content using the BCA kit method. Phosphorylated tyrosine-1175 (rabbit anti-p-tyr1175 VEGFR-2, Cell Signaling Technology) and total VEGFR-2 (rabbit anti-VEGFR-2, Cell Signaling Technology) levels were analyzed by immunoblotting.
Assessment of the effect of CS on VEGF-induced cell migration (scratch assay)
The "scratch assay" was performed to investigate the effect of CSE on VEGF-induced endothelial cell migration. HMVEC-Ls were grown (90% confluent) in 60 mm dishes and starved for 6 h in 0.1% serum and VEGF-free media. Cells were then treated for 2 h with CSE (0.25%), N-acetyl-L-cysteine (NAC; 1 mM) + CSE (0.25%), VEGFR-2 inhibitor (1 µM), and NAC (1 mM) + VEGFR-2 inhibitor (1 µM). NAC treatment was performed 1 h before CSE treatments. At the end of the 2 h, the bottoms of the culture plates were scratched using a 200 µl pipette tip and treated with VEGF (20 ng/ml). Parallel control experiments were carried out without VEGF treatments. The number of cells that migrated beyond the scratch lines after 12 h was counted under the microscope by a single investigator in a blinded manner.
Assay of the effect of CS on VEGF-induced angiogenesis (capillary-like tube formation assay)
To determine the effect of CSE on VEGF-induced tube formation, cells were starved for 6 h in EGM-2 media without VEGF containing 0.1% serum and treated with CSE (0.25%) or VEGFR-2 inhibitor (NVP-AAD777; 1 µM) for 2 h. After 2 h, HMVEC-Ls were washed and plated in 24-well plate that had been precoated with 100 µl of growth factor reduced Matrigel Matrix (BD Bioscience) and cultured at 37°C in a humidified incubator containing 5% CO2 for 24 h with or without 20 ng/ml of VEGF. At the end of 24 h, capillary-like tubes were photographed under a microscope and all side branches were counted by a single investigator in a blinded manner.
Statistical analysis
The Sigma Stat 3.0 statistical program was used to analyzed the data. The results are shown as the mean ± SE. All the data were tested for the normality, and equal variance test to determine whether our sample size is appropriate to analyze the data using ANOVA. If the equal variance or normality test was failed, the data were analyzed using nonparametric analysis (Kruskal-Wallis test). All pairwise multiple comparisons were performed using the Student-Newman-Keuls method; values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, B). The numbers of macrophages in the lung tissue sections were counted after immunostaining was performed with a Mac-3 antibody. The number of macrophages in the lung sections was significantly increased in both CS-exposed groups with and without VEGFR-2 inhibitor treatments compared with their respective air-exposed groups (P<0.05). Furthermore, the number of macrophages in the VEGFR-2 inhibitor-treated CS-exposed group was increased significantly compared with vehicle-treated CS-exposed group (P<0.05; Fig. 1C, D
). The number of neutrophils was also counted in the lung sections after immunostaining was performed with an antibody specific for neutrophils. There was no significant change in the number of neutrophils in the lungs with different treatment groups (data not shown). PEG-400 was used as a vehicle control, and no effect of PEG-400 was observed on any parameters measured. Overall, these data suggested that inhibition of VEGFR-2 augmented CS-induced macrophages influx into the mouse lung.
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CS and VEGFR-2 inhibitor increased proinflammatory mediators in mouse lung
The effects of CS and VEGFR-2 inhibitor on the levels of proinflammatory mediators (KC, MCP-1, IL-6, and TNF-) in mouse lung homogenates were assessed using a Luminex beadlyte assay kit and were normalized with protein levels in lung homogenates. The levels of KC were significantly increased in the vehicle-treated CS group and both the VEGFR-2 inhibitor-treated air and CS groups compared with the vehicle-treated air group (P<0.001). Furthermore, KC levels were significantly increased in the VEGFR-2 inhibitor-treated CS-exposed group compared with the vehicle-treated CS-exposed groups (P<0.05), suggesting that VEGFR-2 inhibition augmented the CS-induced KC levels in mouse lung. Although no increase in MCP-1 level was observed in vehicle-treated CS-exposed group, a synergistic increase in MCP-1 level was observed in the VEGFR-2 inhibitor-treated CS-exposed group (P<0.05). The levels of IL-6 and TNF- in the lung homogenates were significantly increased in both the vehicle and VEGFR-2 inhibitor-treated CS-exposed groups compared with their respective air groups (Fig. 2
). However, the synergistic effects of CS exposure + VEGFR-2 inhibition was not observed for IL-6 and TNF- release. Overall, these data showed that inhibition of VEGFR-2 augmented CS-induced release of key proinflammatory mediators in mouse lung.
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4-HNE adducts levels were increased in response to CS exposure and VEGFR-2 inhibition in mouse lung
CS exposure and VEGFR-2 inhibitor-mediated oxidative stress response were investigated by assessing 4-HNE adducts levels by immunohistochemistry in mouse lung sections. 4-HNE is a stable end product of lipid peroxidation in the cells. Higher levels of 4-HNE are found in the cells during oxidative stress due to increased lipid peroxidation, and therefore, it is used as a marker to assess oxidative stress. The 4-HNE adduct levels (staining score) were significantly increased in both airway and alveolar cells of the lung sections of mice treated with either VEGFR-2 inhibitor (P<0.001) or exposed to CS (P<0.001) or a combination of both(P<0.001) compared with vehicle-treated air group (Fig. 3
A, B). Furthermore, the 4-HNE adducts level (staining score) were significantly increased in combined VEGFR-2 inhibitor-treated CS-exposed group compared with CS exposure alone group in airway epithelial cells (P<0.01). These data indicated that CS exposure or VEGFR-2 inhibitor treatment alone induces oxidative stress and in combination of VEGFR-2 inhibitor treatment and CS exposure oxidative stress was further increased particularly in airway epithelial cells.
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ROS production was increased in response to CSE and VEGFR-2 inhibition in vitro in HMVEC-Ls
CSE- and VEGFR-2 inhibitor-induced ROS production were investigated using the fluorescent dye carboxy-H2DCF-DA in vitro utilizing HMVEC-Ls and confirmed the data of oxidative stress effect of VEGFR-2 inhibitor in mouse lung. ROS production was significantly increased by CSE treatments in a concentration-dependent manner in HMVEC-Ls (data not shown). VEGFR-2 inhibitor alone caused significantly increased ROS production compared with control experiments in HMVEC-Ls (P<0.01). Furthermore, ROS production was significantly increased when the cells were treated with CSE (0.25%) in combination with the VEGFR-2 inhibitor (0.5 µM) compared with CSE (0.25%) or VEGFR-2 inhibitor treatments alone in HMVEC-Ls (P<0.05; Fig. 3C, D
). These data showed that both CSE and VEGFR-2 inhibition caused increased ROS production in HMVEC-Ls.
VEGFR-2 levels and its phosphorylation were decreased in response to CS and/or VEGFR-2 inhibition in mouse lung
The effects of CS and VEGFR-2 inhibitor on tyrosine-phosphorylated VEGFR-2 levels in mouse lungs were assessed by immunoblotting. The levels of phosphorylated VEGFR-2 were normalized with total VEGFR-2 found on the same blot. Significantly decreased levels of phosphorylated VEGFR-2 were observed in mouse lung tissues of the VEGFR-2 inhibitor-treated groups compared with vehicle groups (P<0.05), suggesting that subcutaneous injection of VEGFR-2 inhibitor (NVP-AAD777) impaired VEGFR-2 phosphorylation in the mouse lung (Fig. 4
A, B). These data also confirmed the effectiveness of the dose of VEGFR-2 inhibitor administered (20 mg/kg body weight). The levels of VEGFR-2 expression were significantly reduced in both VEGFR-2 inhibitor-treated air- and CS-exposed groups and the vehicle-treated CS-exposed group compared with vehicle-treated air group (P<0.05; Fig. 4A, C
). These data revealed that both CS and VEGFR-2 inhibition caused down-regulation of VEGFR-2 receptor protein expression in mouse lung.
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CSE down-regulated VEGFR-2 expression and impaired VEGF-induced VEGFR-2 phosphorylation in HMVECs
Because VEGFR-2 is an essential mediator of VEGF-induced endothelial function, the effect of CSE on VEGFR-2 expression level and VEGF-induced VEGFR-2 phosphorylation was assessed in vitro in HMVEC-Ls. We investigated the effect of CSE on basal VEGFR-2 protein expression level in a time- and concentration-dependent manner. Our data showed that at 2 h of CSE treatments, VEGFR-2 protein levels were neither down-regulated nor phosphorylated, but at 12 h of CSE treatment, VEGFR-2 levels were down-regulated. Therefore, to determine whether CSE had any effect on VEGF-induced VEGFR-2 phosphorylation, the 2 h time point was selected so as to rule out the direct effect CSE on VEGFR-2 down-regulation. The levels of total and phosphorylated (tyrosine 1175) VEGFR-2 were measured by immunoblotting after the cells were treated with VEGF. VEGFR-2 phosphorylation was significantly decreased in CSE-treated cells in concentration-dependent manner (0.1–0.5%) compared with the control experiment (P<0.05; P<0.01; Fig. 5
A, B). These data revealed that the CSE concentration dependently down-regulated VEGF-induced VEGFR-2 phosphorylation in vitro in HMVEC-Ls. Furthermore, it is confirmed that VEGF-induced VEGFR-2 phosphorylation was completely inhibited by the VEGFR-2 inhibitor NVP-AAD777 at the concentration of 1.0 µM in vitro in HMVEC-Ls. DMSO vehicle treatment had no effects on VEGF-induced VEGFR-2 phosphorylation. To determine the effect of CSE on VEGFR-2 expression levels, HMVEC-Ls were starved and treated for 12 h, and VEGFR-2 levels were measured by immunoblotting. CSE concentration dependently (0.1–0.5%) down-regulated the VEGFR-2 expression levels (P<0.05; Fig. 5C, D
). These new finding showed that CSE concentration dependently down-regulated the levels of VEGFR-2 in vitro in HMVEC-Ls.
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VEGF levels were decreased in response to CS exposure and/or VEGFR-2 inhibition in mouse lung
To investigate the effect of CS and VEGFR-2 inhibitor on VEGF expression, VEGF protein levels were assessed by a Luminex beadlyte kit and normalized with protein levels in mouse lung homogenates. The VEGF levels in mouse lung were significantly decreased in the vehicle-treated CS-exposed group and VEGFR-2 inhibitor-treated air and CS-exposed groups when compared with the vehicle-treated air group (P<0.05; P<0.01). Further decreases in VEGF levels were observed in combination of the VEGFR-2 inhibitor- and CS-exposed group compared with vehicle-treated CS-exposed group in mouse lungs, suggesting that VEGFR-2 inhibition caused further decreased of CS-induced down-regulation of VEGF (P<0.05). These data suggested that both CS and VEGFR-2 inhibition led to down-regulation of VEGF protein expression in mouse lungs (Fig. 6
).
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Phosphorylated and total eNOS protein levels were decreased in mouse lungs in response to CS and/or VEGFR-2 inhibition
VEGF-induced VEGFR-2 phosphorylation and downstream signaling induce activation of eNOS, a key enzyme linked to endothelial function. Therefore, the effects of CS exposure and VEGFR-2 inhibition on p-eNOS and total eNOS protein levels were assessed in mouse lung. Our data showed that CS exposure or VEGFR-2 inhibition significantly reduced the level of p-eNOS and total eNOS protein in mouse lung as compared with that of the vehicle-treated air-exposed group (Fig. 7
). Furthermore, inhibition of VEGFR-2 augmented CS-induced reduction of p-eNOS levels in mouse lung (P<0.05). These data suggested that eNOS is more susceptible to the effect of CS when its signaling is impaired by VEGFR-2 inhibition in mouse lung.
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CS down-regulated the levels of eNOS in vitro in HMVEC-Ls
Since VEGFR-2 and eNOS are essential mediators of VEGF-induced endothelial function, the effect of CSE on eNOS protein levels in vitro in HMVEC-Ls was determined. Cells were starved and treated for 12 h with CSE, and eNOS protein levels were measured by immunoblotting method. CSE concentration dependently (0.25 and 0.5%) down-regulated the eNOS protein levels (P<0.05; Fig. 8
A, B). However, the levels of eNOS were unaffected after 12 h at a relatively low level of CSE (0.1%). Nevertheless, these data suggested that CSE down-regulated the eNOS protein levels in HMVECs.
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CS impaired VEGF-induced cell migration and angiogenesis in vitro in HMVEC-Ls
We further investigated whether the CSE-mediated decrease in the level of VEGFR-2 and VEGF-induced VEGFR-2 phosphorylation caused impaired cell migration and angiogenesis using the scratch and capillary-like tube formation assays, respectively. Specific VEGFR-2 inhibitor, NVP-AAD777 was used to compare the effect of CSE-mediated VEGF-induced cell migration and angiogenesis. VEGF-induced cell migration (P<0.01; Fig. 9
A, B) and capillary-like tube formation (P<0.05; Fig. 9C, D
) were significantly reduced in CSE-treated HMVEC-Ls compared with control (VEGF-treated) experiments. Furthermore, pretreatment of HMVEC-Ls with NAC, a thiol antioxidant, significantly attenuated the CSE-induced impaired cell migration (P<0.05). However, VEGFR-2 inhibitor-mediated VEGF-induced impaired cell migration was not reversed by NAC pretreatment. These data suggested that CSE-induced impaired cell migration is due to ROS generated from CSE or the presence of electrophilic compounds in CSE. Significantly inhibited cell migration and capillary-like tube formations were observed in HMVEC-Ls treated with VEGFR-2 inhibitor, demonstrating the involvement of VEGF-induced VEGFR-2 phosphorylation and its downstream signaling pathway in endothelial cell migration and angiogenesis (P<0.001). In both scratch and tube formation assays, we observed that VEGFR-2 inhibition (2 h) prevents attachment of cells to the culture plates and some morphological changes. However, cell viability was not significantly affected with VEGFR-2 inhibition as assessed by trypan blue exclusion test.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 25
, 29)
. Our results confirmed that CS induced an influx of inflammatory cells and increased proinflammatory mediators (KC and MCP-1) in mouse lungs. Surprisingly, inhibition of VEGFR-2 augmented CSE-induced inflammatory response in the lung. However, synergistic effects of CS exposure and VEGFR-2 inhibition were not seen for IL-6 and TNF- release. It is known that IL-6 and TNF- act as autocrine growth factors for tumor angiogenesis and that these cytokines can be prometastatic or proangiogenic (30
, 31)
. It is also known that inhibition of angiogenesis down-regulated the levels of IL-6 and TNF- in mouse serum (31)
. Since the VEGFR-2 inhibitor (NVP-AAD777) is antiangiogenic, it is possible that inhibition of VEGFR-2 may have a confounding effect on these cytokine release in mouse lung. The other reason may be that the there was already a threshold level of induction of IL-6 and TNF- in response to CS exposure, which was not augmented by VEGFR-2 inhibition. Nevertheless, the number of macrophages in lung interstitium and the levels of MCP-1 and KC in lung tissue was significantly increased in the group treated with VEGFR-2 inhibitor and subsequent exposure to CS, suggesting that VEGFR-2 inhibition augmented the CS-induced inflammation in mouse lungs.
Oxidative stress was increased with VEGFR-2 inhibition and CS exposure, and this was augmented in combination of VEGFR-2 inhibition with CS exposure as assessed by lipid peroxidation products (4-HNE adducts) stained in airway epithelial cells in vivo in mouse lung and carboxy-H2DCF-DA fluorescent staining (ROS release) in vitro in HMVEC-Ls. However, the mechanism for VEGFR-2 inhibitor induced ROS generation and inflammation in the lung is not clear. Increased levels of oxidative stress, as measured by 4-HNE adducts staining, occur in the lungs of smokers and in patients with COPD (32)
. It was also shown that chronic inhibition of global VEGFR by SU5416 resulted in oxidative stress in rat lungs (23)
. It was speculated that the interruption of VEGF survival signals is interpreted by lung cells as a "danger signal" resulting in oxidative stress as a downstream response (23)
. Hence, we propose that the mechanism of VEGFR-2 inhibitor-induced oxidative stress in vivo in mouse lung and in vitro in HMVEC-Ls may either be due to activation of NADPH oxidase or dual oxidases in endothelial cells; however, this concept requires further investigation.
Both VEGF and VEGFR-2 expression are decreased in emphysematous lungs (20
, 21)
. VEGF levels in the BALF were also decreased in smokers (33)
but not in the lungs of patients with mild COPD (34
, 35)
. It is also interesting to note that chronic inhibition of VEGFR leads to enlargement of airspaces and decreased capillary density, which is characteristic of emphysema in rat lungs (21
, 36)
. Moreover, patients with COPD also display reduced vascularization of the lung, including decreased capillary density (37)
. In the present study, we showed the decreased levels of VEGF and VEGFR-2 levels in both the VEGFR-2 inhibitor and CS-exposed groups in vivo in mouse lung and the decreased level of CSE-mediated VEGFR-2 expression in vitro in HMVEC-Ls. These results corroborate previous findings in smokers and in patients with COPD showing a decreased lung level of VEGF and VEGFR-2 (22)
and implicate CS-derived free radicals as being directly involved in down-regulation of VEGF and VEGFR-2 levels in endothelial cells. However, the mechanism of CS-mediated down-regulation of VEGF and VEGFR-2 is not known. It is possible that aldehyde-mediated modifications of cellular signaling may impair VEGF release from epithelial cells and macrophages as shown by 4-HNE adducts formation in these cells in response to NVP-AAD777 treatment and CSE exposure. CS exposure may also alter kinase signaling mechanism or induce post-translational modifications of VEGFR-2 subsequently leading to its degradation.
VEGF phosphorylates VEGFR-2, and its downstream signaling activates eNOS, which is a key enzyme in the regulation of endothelial cell migration, proliferation, and survival (1)
. Cigarette smoking is associated with endothelial dysfunction (2
, 8
, 10
11
12)
. Clinical data also showed impaired vascularization in smokers and those with COPD (38)
. However, the exact mechanisms involved in CS-induced endothelial dysfunction and decreased vasculature in the lungs are not known. Hence, we propose that CS causes down-regulation of VEGFR-2 expression and impairs VEGF-induced VEGFR-2 phosphorylation and eNOS activation resulting in endothelial dysfunction. To support these observations, we carried out in vitro experiments using HMVEC-Ls to determine the CSE-mediated down-regulation of VEGFR-2 levels. It is known that phosphorylation of VEGFR-2 tyrosine residue at 1175 (in human) is essential for vasculogenesis and inhibition of phosphorylation may be associated with reduced VEGFR-2 downstream signaling (39)
. Consistent with in vivo findings, CSE down-regulated VEGFR-2 and eNOS protein expression and VEGF-induced VEGFR-2 tyrosine (tyr-1175) phosphorylation in endothelial cells. Similarly, decreased levels of phosphorylated VEGFR-2 were shown in the lungs of smokers and COPD patients (22)
. The reason for CS-mediated down-regulation of VEGFR-2 phosphorylation is not known. However, our data indicated that tyrosine residues are nitrated (unpublished results) in response to CSE treatment rendering VEGFR-2 inactive for downstream kinase signaling or affecting its activity. The other likely reason would be that CS-derived lipid peroxidation products, such as 4-HNE and acrolein and/or reactive nitrogen species, react with VEGFR-2 rendering VEGFR-2 inactive for downstream kinase signaling or affecting its activity (40
, 41)
.
VEGF-mediated VEGFR-2 phosphorylation and downstream signaling induce eNOS activation in endothelium (1)
. Since eNOS is an essential mediator of VEGF-VEGFR-2-mediated endothelial function, we determined whether CS exposure had any effect on phosphorylation of eNOS and its levels in mouse lung in vivo and in HMVEC-Ls in vitro. VEGFR-2 inhibition led to CS-induced down-regulation of phosphorylated eNOS and total eNOS both in HMVEC-Ls and mouse lung. However, at low levels of CSE the eNOS protein level was not affected in vitro in HMVEC-Ls. It has been shown that CSE causes phosphorylation of eNOS via activation of redox-sensitive phosphoinositide 3-kinase (PI3K) and Akt-signaling pathways (42)
. Furthermore, phosphorylation of eNOS released NO and up-regulated the expression of eNOS protein (43)
. Our data showed no effect for low concentration of CSE on eNOS levels, suggesting a VEGFR-2-independent (bypassing VEGFR-2) signaling mechanism possibly by direct activation PI3K and Akt is responsible for nullifying the effect of CSE-induced eNOS down-regulation in endothelial cells in vitro. However, at high concentrations of CSE, the PI3-kinase/Akt-induced eNOS activation is impaired by CSE-mediated aldehyde/oxidative stress-induced effects, which would then impair VEGFR-2-mediated downstream signaling for eNOS phosphorylation. Indeed, Su et al. (44
, 45)
have shown CSE (high concentrations) mediated irreversible inhibition of eNOS activity in endothelial cells, possibly by kinase signaling mechanisms. Nevertheless, our in vivo data clearly show down-regulation of phophorylated eNOS, suggesting CS-mediated endothelial dysfunction in the lung.
It has previously been shown that NO donors can rescue cellular migration and tube formation in endothelial cells exposed to CSE, suggesting that impaired NO biosynthesis is related to dysfunctional endothelium-dependent vasodilation in smokers (19)
. We further investigated whether the CSE-mediated decrease in the level of VEGFR-2 and VEGF-induced VEGFR-2 phosphorylation caused impaired cell migration and angiogenesis. Both angiogenesis and cell migration are important in neovascularization in the lung. Angiogenesis is driven by microvascular endothelial cells, which on activation by angiogenic growth factors such as VEGF migrate into the interstitial matrix, proliferate, and form new capillary-like structures (46)
. Our data showed that CSE impaired endothelial cell migration and angiogenesis as assessed by wound healing scratch and capillary-like tube formation assays, respectively. Hence, the endothelial dysfunction seen in smokers could possibly be related to an impaired response to VEGF. The CSE-mediated inhibition of endothelial cell migration may be associated with a reduced expression of several integrins that are involved in NO-dependent endothelial migration (1)
. The signaling mechanisms for CS-induced alteration in cell migration and angiogenesis are not known. We hypothesize that CS-induced ROS and free radicals (micromolar to millimolar levels) cause alterations in VEGF-VEGFR-2 downstream signaling for cell migration and angiogenesis. This may be the mechanism for CS-induced endothelial dysfunction. However, it has been shown that VEGF signaling is mediated via NADPH oxidase-derived ROS production (nanomolar to picomolar levels) in particular by the gp91phox subunit of NADPH oxidase leading to downstream signaling of angiogenesis (47
, 48)
. Hence, a fine tuning exists between low levels of ROS, which is proangiogenic, and high levels of ROS and free radicals and aldehydes, which may be antiangiogenic.
Our data showed that pretreatment of HMVEC-Ls with NAC, a thiol nucleophilic antioxidant, significantly attenuated the CSE-mediated VEGF-induced impaired cell migration. These findings suggested that CSE-mediated impaired cell migration is associated with oxidative stress imposed by CSE. We further observed that CS-inhibited VEGF-induced phosphorylation of VEGFR-2 was reversed by NAC, suggesting that NAC is in fact conjugating free radicals and aldehydes present in CS (unpublished results). Similarly, Michaud et al. (19)
have recently shown that CSE-impaired VEGF-induced endothelial cell migration and tube formation was free radical dependent. However, VEGFR-2-inhibited cell migration was not reversed by NAC pretreatment, although VEGFR-2 inhibition was associated with ROS generation. The differential effect of NAC on CSE- and VEGFR-2 inhibitor-mediated attenuation of cell migration is not known. It is possible that ROS production in response to VEGFR-2 inhibition is secondary to its ability to affect cell migration. Nevertheless, these data suggest that CSE-induced (but not VEGFR-2-induced) impaired cell migration is due to either ROS generated from CSE (via NADPH oxidase activation) or to the electrophilic compounds present in CSE (49)
.
Taken together, our data of mouse lung in vivo and in human endothelial cells in vitro support the concept that inhibition of VEGFR-2 led to increased oxidative stress and inflammatory response and CS exposure further augmented oxidative stress and inflammatory response. Overall, CS-induced oxidative stress impaired both VEGF-induced VEGFR-2 phosphorylation and VEGFR-2 expression in HMVEC-Ls in vitro and in mouse lungs in vivo leading to endothelial dysfunction as assessed by decreased levels of eNOS, cell migration, and angiogenesis. This, in turn, could lead to endothelial dysfunction associated with the development of atherosclerotic lesions and the triggering of acute ischemic events in patients exposed to CS. Our data also demonstrate that the CS-induced impaired endothelial function was attenuated by pretreatment with the thiol antioxidant NAC in HMVEC-Ls in vitro. These new findings not only define the basic understanding of oxidant-induced endothelial dysfunction via the VEGF-VEGFR-2 specific pathway but also have broad implications in the pathogenesis of various CS-mediated pulmonary and cardiovascular diseases where endothelial dysfunction occurs. Further studies are in progress to determine the mechanisms by which CS mediates VEGF-VEGFR-2-induced endothelial dysfunction in the lung.
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ACKNOWLEDGMENTS |
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Received for publication October 11, 2007. Accepted for publication January 10, 2008.
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